Developers of therapeutic biologic-based pharmaceuticals seek to minimize potential immunogenicity thereof, for example, by humanization, cloaking immunogenic sites or developing chimeric constructs by combining alleged minimally immunogenic or non-immunogenic human sequences with active domains from, for example, non-human sources which are potentially immunogenic. However, in spite of those measures, such drugs or composite drugs remain immunogenic or the potential remains for patients to develop an immune reaction to the administered drug, rendering the drug initially ineffective, progressively ineffective and/or life-threatening, because of, for example, an anaphylactic reaction thereto.
It is therefore important to measure the immunogenicity of drugs, for example, during the development process to optimize drug design. Also, such assays can be used to determine the responsiveness of a recipient to a drug, either prior to exposure or to monitor a patient during a course of treatment. Thus, an assay that detects anti-drug antibodies (ADA), for example, in the serum or plasma of host animals or patients undergoing treatment, can be used for those reasons.
Some current methods for measuring drug immunogenicity employ enzyme-linked immunosorbent assay (ELISA) materials and methods. Those assays generally utilize a double antigen bridging format wherein the drug is coated onto a solid phase, such as, a well in a microtiter plate, and a labeled version of the same drug is dissolved in a liquid medium. The label can consist of a signal-developing enzyme or a binding moiety that can complex with a binding partner labeled with a signal-developing enzyme. The ADA in the patient sample can then form a bridge complex wherein one binding site attaches to the coated antigen and another binding site binds the labeled drug. After washing and addition of substrate reagents, the wells containing ADA will develop a signal associated with the bound enzyme.
Surface plasmon resonance (SPR) assays are also used for the determination of immunogenicity using a direct and label-free assay format.
But those assays suffer from being labor-intensive and time-intensive as well as requiring complex and expensive equipment. In some cases, a sample dilution step is required to reduce background (i.e., for SPR and ELISA assays) or multiple washing steps are needed to separate bound from free antibodies and antigens (i.e., for ELISA). The washing step(s) can remove low affinity and low avidity ADA, for example, as antibodies have different on-rates and off-rates, detection of antibodies with fast off rates may not be detected due to extensive washing. Further, the sample dilution step can result in the identification of a negative response when patients may actually possess low, but clinically relevant, levels of ADAs.